Construction, Expression, and Purification of p28 as a Cell-Penetrating Peptide ‎with Anticancer Effects on Burkitt’s Lymphoma Cell Line

The p28 is a small-sized cell-penetrating peptide derived from bacterial protein azurin and can function as a cancer-specific anti-proliferative agent. It can penetrate cancer tissues easily without involving the immune system, and increase the intracellular concentration of p53. In this study, we have expressed and purified recombinant p28, then evaluated its anti-proliferative and pro-apoptotic effects on Raji cancer cell line. The p28 gene was amplified and cloned into pTZ57R cloning vector and was sequenced subsequently. Afterward, it was transformed into E. coli BL21 bacterial host by using pET-28a expression vector. Peptide purification was carried out using Ni-NTA ‎chromatography system. Bradford, SDS-PAGE, and western blotting assays were applied to assess the concentration and expression level of the recombinant peptide. The proficiency ‎of p28 in inhibition of tumor growth and induction of apoptosis in cancerous cells was investigated by evaluating the Raji and HEK-293 cells treated with different concentrations of p28. The overexpression of the p28 peptide in the bacterial host was confirmed by SDS-PAGE and western blotting. Moreover, Bradford assay revealed desirable concentrations of the recombinant p28 before and after dialysis. The MTT and PE-Annexin V apoptosis assays indicated the specific function of p28 in impeding the proliferation of cancerous cells and triggering the apoptosis. The p28 induces apoptosis in cancerous cells but not in normal control cells. ‎In summary, p28 is a non-immunogenic small peptide that can penetrate cancerous cells ‎preferentially, impede the cell proliferation, and induce the apoptosis. Overall, these ‎findings suggest p28 as‎ a promising anticancer drug.